This is the third renewal application for this project to understand the pathophysiology of proteinuric kidney diseases. Progress over the last 20 years has focused attention on the podocyte, at least in part because of our pioneering work on CD2AP. In the last funding period, we hypothesized that foot process effacement, a universal feature of proteinuria, was the physiological manifestation of Rac activation. Previous in vitro studies showed that Rac controlled actin cytoskeletal structures like the lamellopodia and membrane ruffling. We postulated that podocytes maintain high levels of Rho activity, which functions to enhance cell adhesion by controlling the formation of actin cables known as stress fibers. To prove our hypothesis, we showed that the acute activation of Rac induced foot process effacement and proteinuria. However, others in the field have argued that it is Rho activation that is the cause of effacement and proteinuria. Because all of this work is based on transgenics, which are difficult to compare because of the number of copies of the transgene and because of random integration, we propose to generate mice using ES cell technology, where a single copy of the transgene is targeted to a specific chromosomal locus. This will allow different transgenics to be compared to each other. In specific aim #1, we will compare and contrast three transgenic mice that we generated that allow for inducible expression of constitutively active Cdc42, RhoA and Rac1. In specific aim #2, we will test whether the activation of RhoA can attenuate the activation of Rac1 and proteinuria in several different models of podocyte injury. Since GPCRs are a major regulator of Rho in cells, and as we believe that Rho activation to be a potential strategy for the treatment of proteinuria, we propose to first characterize all of the GPCRs expressed in podocytes. We will then test whether ligands for this GPCRs can activate Rho using both cultured podocytes and a novel minced kidney assay that we developed in the last funding period. We believe that a better understanding of the mechanistic cell biological causes of foot process effacement and proteinuria will allow for novel therapeutics to be developed that will allow this process to be reversed.